Biostimulants alleviate water deficit stress and enhance essential oil productivity: a case study with savory

Water deficit stress exposure frequently constrains plant and agri-food production globally. Biostimulants (BSs) can be considered a new tool in mitigating water deficit stress. This study aimed to understand how BSs influence water deficit stress perceived by savory plants (Satureja hortensis L.), an important herb used for nutritional and herbal purposes in the Middle East. Three BS treatments, including bio-fertilizers, humic acid and foliar application of amino acid (AA), were implemented. Each treatment was applied to savory plants using three irrigation regimes (low, moderate and severe water deficit stress FC100, FC75 and FC50, respectively). Foliar application of AA increased dry matter yield, essential oil (EO) content and EO yield by 22%, 31% and 57%, respectively. The greatest EO yields resulted from the moderate (FC75) and severe water deficit stress (FC50) treatments treated with AA. Primary EO constituents included carvacrol (39–43%), gamma-terpinene (27–37%), alpha-terpinene (4–7%) and p-cymene (2–5%). Foliar application of AA enhanced carvacrol, gamma-terpinene, alpha-terpinene and p-cymene content by 6%, 19%, 46% and 18%, respectively. Physiological characteristics were increased with increasing water shortage and application of AA. Moreover, the maximum activities of superoxide dismutase (3.17 unit mg−1 min−1), peroxidase (2.60 unit mg−1 min−1) and catalase (3.08 unit mg−1 min−1) were obtained from plants subjected to severe water deficit stress (FC50) and treated with AA. We conclude that foliar application of AA under water deficit stress conditions would improve EO quantity and quality in savory.


Materials and methods
Experimental area. Two field experiments were conducted in 2020 and 2021 at a farm in the Higher Education Center of Shahid Bakeri Miandoab, Iran. Weather (mean temperature and annual precipitation) and soil physicochemical characteristics (taken at the 0-30 cm depth) of the experimental area are shown in Tables 1 and 2, respectively.

Plots preparation, treatments and cultivation. The experiments were established in a randomized
complete block design (RCBD) with a split-plot arrangement and three replicates. Each subplot was composed of 10 rows of savory with an inter-row spacing of 40 cm. There was a 25 cm distance between plants within the rows. The subplots were 2.5 × 4 m. Plots and blocks were separated by a buffer space of 2 m and 3 m, respectively. Main plots were allocated to different irrigation regimes (3 levels), including full irrigation (FC100), moderate (FC75) and severe water stress (FC50), while sub-plots were assigned to different fertilizer sources (4 levels), including no fertilization (control), humic acid (HA), bio-fertilizer (B) bacteria [mixture of phosphate-solubilizing bacteria (PSB) Pantoea agglomerans + Pseudomonas putida, K-solubilizing bacteria (KSB) Pseudomonas koreensis + Pseudomonas vancouverensis, and N-fixing bacteria (NFB) Azotobacter vinelandii] and foliar application of amino acid (AA).
The savory (Miandoab local landraces) seeds were obtained from Urmia University, Iran. For bio-fertilizer treatments, savory seeds were soaked in biofertilizer agents (10 8 active bacteria per g) and dried in the shade for  www.nature.com/scientificreports/ one h before seeding. Seeds were manually sown on May 4, 2020, and May 6, 2021. There was a 25 cm distance between plants within the rows. Weeds were regularly removed by hand when needed. Each experimental unit utilised a total application rate of 200 mg of the HA powder for the HA treatment. One-third of this amount was dissolved in water and applied at seed sowing, while the remaining two-thirds were added to the water and used at the stem elongation and later at the flowering stage. The HA treatment was added in the respective experimental units with the first irrigation (5-10 L ha −1 ; after seed sowing and before flowering). The HA treatment (Humax® 95-WSG) contained 80% humic acid, 15% folic acid, and 12% potassium. Foliar application of the AA treatment (43.7% total protein, 50% amino acids, and 20% total N) was performed twice in each growing season, once at the beginning of the shooting stage and once four weeks later at the concentration of 2 L per 1000 L of water. Spraying was done in the afternoon near sunset. Control plants were sprayed using distilled water.
The plant material and seeds were obtained under the supervision and permission of Urmia University and according to national guidelines, and all authors comply with all the local and national guidelines.
Measurements. Agronomic traits. Ten plants were randomly selected from each plot at the full flowering stage to determine agronomic traits, including plant height, canopy diameter, and lateral branch number. For dry matter determination, a 1.6 m 2 area was harvested at each plot on August 10, 2020, and August 14, 2021.
Essential oil extraction and analysis. The savory EO was extracted using the water distillation method by a Clevenger apparatus. For this, 40 g of dried plant material (leaves + flowers) was added to the balloon of the essential oil extraction machine and 500 ml of distilled water was added. The essential oil extraction operation was carried out for three hours. The extracted EO was dried with anhydrous sodium sulfate and kept at 4 °C for further chemical analysis. EO yield (kg ha −1 ) was calculated by multiplying the dry matter yield by its EO percentage. Oil constituents were analyzed using a GC-MS (Agilent 7890/5975A), as suggested by Namazi et al. 27 .
Relative water content. At the flower initiation stage, the leaves' relative water content (RWC) was determined according to Levitt 28 , as follows: where F w , T w, and D w represent the fresh, turgid (by floating the fresh leaves in double distilled water at 4 °C for 5 h) and dry weight of leaves (by drying the leaves in an oven at 70 °C for 48 h), respectively.
Physiological characteristics. In the flowering stage, 0.5 g fresh savory leaves were homogenized in 10 mL of 80% acetone and centrifuged at 12,000 rpm for 15 min. After that, the absorbance was read at 663 nm, 645 nm and 470 nm by a UV spectrophotometer (UV-1800, Shimadzu, Tokyo, Japan). The content of chlorophyll a, b and carotenoids was calculated based on the following equations 29 : In these equations, Chl a, b and Car represent chlorophyll a, b and carotenoid concentrations, respectively. Parameters A646.8, A663.2 and A470 represent the absorbance measured at 646.8, 663.2 and 470 nm using a spectrophotometer.
Proline concentration was determined following the method described by Bates et al. 30 . First, 0.5 g of fresh leaves were homogenized with 10 mL of 3% sulfosalicylic acid and centrifuged at 12,000g for 10 min at 4 °C. Then, 2 mL of each sample was boiled with 2 mL of acid-ninhydrin + 2 mL of glacial acetic acid for 40 min. The reaction mixture was extracted with 4 mL of toluene and mixed with a vortex for 20 s. Finally, the absorbance was measured spectrophotometrically at 520 nm compared to the clean toluene. The soluble sugar concentration (SSC) was determined using the phenol and sulfuric acid method. Briefly, 0.5 g of savory fresh leaves were homogenized with ethanol and mixed with 98% sulfuric acid and 5% phenol and absorbance were determined with a spectrophotometer at 485 nm 31 .
First, to assess the antioxidant enzyme activity across different irrigation levels and fertilizer sources, 0.1 g of savory leaves (frozen in liquid nitrogen) were homogenized in 2 mL sodium phosphate buffer and centrifuged at 15,000 rpm at 4 °C for 20 min. Then, the supernatant was used to determine antioxidant enzyme activity. The superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) activities were measured according to the procedures reported by Dhindsa et al. 32 , Kar and Feierabend 33 and Aebi 34 , respectively. All enzyme activity was reported in units of mg g −1 min −1 .
Antioxidant activity. The 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) assay was utilized to determine the plant's antioxidant activity. For this purpose, 3.8 mg of DPPH was dissolved in 25 mL MeOH. Next, 100 µL of the mixture was added into individual wells of 96-well plates and mixed with 85 µL MeOH and 15 µL of savory sample extract. Finally, the microplates were incubated in the dark and at room temperature for 30 min, and the absorbance was read at 515 nm 35 . Duncan's multiple range test at a 5% probability level were performed using the SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). The irrigation levels and fertilizer sources were considered fixed effects, while block, year, and interactions were considered random. Correlation coefficients among all variables under analysis were also calculated using the SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). The heatmap clusters were created in Ward's method using heatmap.2 functions in gplots R-package.

Results
The analysis of variance in Tables 3 and 4 show that the main effects of "Irrigation" (I) and "Fertilizer" (F) were significant for all the agronomical (Table 3) and physiological (Table 4) traits under analysis. However, the interaction between both factors was also significant in all cases, so this is the effect that was analyzed for all traits under study.
Plant height. The maximum plant height (68.7 cm) was obtained from FC100 treatment fertilized with AA followed by HA application (67.8 cm). However, the minimum plant height (46.8 cm) was achieved from FC50 treatment without fertilization (control). Compared with FC100, plant height decreased in FC75 and FC50 by 10% and 20%, respectively. In addition, plant height was enhanced by 10%, 7% and 14% due to HA, B and AA application (Fig. 1A).
Lateral branch number. The maximum lateral branch number (22.2) was obtained from FC100 treatment fertilized with AA. In contrast, the minimum lateral branch number (12.2) was related to FC50 treatment without fertilization (control). Lateral branch numbers decreased in FC75 and FC50 by 10% and 22%, respectively. Moreover, lateral branch numbers increased by 25%, 19% and 35% due to HA, B and AA applications (Fig. 1B). Dry matter yield (DMY). The maximum (5225 kg ha −1 ) and minimum (2767 kg ha −1 ) DMY was achieved from FC100 treatment fertilized with AA and FC100 without fertilization. The DMY decreased by increasing water deficit stress severity. Compared to the FC100 treatment, DMY decreased in FC75 and FC50 treatments by 11% and 33%, respectively. In addition, fertilization with HA, B and AA increased canopy diameter by 14%, 11% and 22%, respectively (Fig. 1D).
Essential oil (EO) content and yield. The EO content extracted from the aerial parts was the highest (1.27%) in the FC50 treatment treated with AA. However, the lowest EO content (0.72%) was recorded in nostress conditions (FC100) without fertilization. Across irrigation regimes, EO content increased by 21% and 45% in moderate (FC75) and severe (FC50) water deficit stress treatments. Also, the EO content increased by 19%, 18% and 31% due to HA, B and AA applications, respectively ( Fig. 2A). In addition, the highest EO yield was recorded in moderate (FC75) and severe (FC50) water stress treatments treated with AA. The EO yield increased on HA, B and AA application by 35%, 29% and 57% compared with the control, respectively (Fig. 2B).
Carotenoid concentration. The carotenoid content was enhanced with increasing water stress levels. The highest carotenoid concentration (1.7 mg g −1 fresh weight) was measured in FC50 treated with AA. Compared with FC100, the carotenoid concentration was enhanced by 22 and 51% in FC75 and FC50 water stress. Also, fertilization with HA, B and AA enhanced carotenoid concentration by 31%, 27% and 49%, respectively, compared with the control (Fig. 3A).
Relative water content (RWC). The maximum (78%) and minimum (46%) RWC percentage values were related to normal irrigation conditions (FC100) treated with AA and severe water deficit (FC50) without fertilization, respectively. The RWC decreased with increasing water stress levels. Compared with FC100, RWC content was reduced by 15% and 29% in moderate (FC75) and severe water stress (FC50), respectively. Additionally, fertilization with HA, B and AA increased RWC by 11%, 10% and 16%, respectively, compared with the control (Fig. 3B).

Soluble sugars concentration (SSC).
The maximum SSC (4.3 mg g −1 fresh weight) was obtained from FC50 fertilized with AA, followed by HA application (4.2 mg g −1 fresh weight). The SSC was enhanced with increasing water stress levels. Compared with FC100, the SSC increased by 64 and 128% in moderate (FC75) and severe water stress (FC50). Additionally, fertilization with HA, B and AA increased the SSC by 26%, 23% and 30%, respectively, compared with the control (Fig. 3C).
The heatmap clustering showed that the irrigation regimes significantly impacted the studied characteristics more than the biofertilizer treatments. All the biofertilizer treatments with the same irrigation regimes were classified into one group. The evaluated morpho-physiological variables clustered into two distinguished clusters: (1) essential oil yield, dry matter yield, plant height, canopy diameter, lateral branch, chlorophyll a and b, and relative water content; (2) carotenoid and soluble sugars contents, proline, POX, SOD, and CAT activities, antioxidant activity and essential oil content. All the classified variables in the first group decreased significantly with increasing water stress. In contrast, the classified variables in the second group significantly increased by increasing water stress (Fig. 5).
The heatmap clustering of the essential oil compounds based on irrigation regimes and biofertilizers indicated higher amounts of alpha-phellandrene, linalool, beta-pinene, alpha-pinene, limonene, thymol, trans-caryophyllene, beta-bisabolene, and spathulenol were detected under normal-irrigation and control treatment of biofertilizer application. In contrast, these essential oil compounds were remarkably decreased under moderate water stress along with amino acid (FC75 + AA) and bacterial (FC75 + B) biofertilizers and severe water stress along with amino acid (FC50 + AA), bacterial (FC50 + B), and humic acid (FC50 + HA) biofertilizers (Fig. 6). In contrast, a higher amount of p-cymene, carvacrol, and gamma-terpinene were observed under moderate and severe water stress. The heatmap clustering shows moderate and severe water stress accompanied by amino acid and bacterial biofertilizers have the highest impact on essential oil metabolism (Fig. 6).

Discussion
Our results show that plant height, lateral branches, canopy diameter, and dry matter yield all decreased due to moderate (FC75) and severe (FC50) water stress. Under water deficit stress conditions, nutrient absorption decreased around the root zone due to lower mobility and rate of mineral diffusion 36 . Water limitations affect cell differentiation, division, elongation and, finally, a reduction in photosynthesis rate and dry matter production 37 . Daszkowska-Golec et al. 38 noted that water stress conditions reduce the entry of carbon dioxide into the closed stomata due to dehydration, reducing photosynthetic materials supply and plant productivity. Similarly, Amani Machiani et al. 39 noted that thyme (Thymus Vulgaris L.) dry matter yield decreased due to moderate and severe drought stress by 27% and 40%, respectively. Also, under drought stress conditions, AA application positively affected the plant yield components and dry matter yield and also decreased the negative impacts of drought stress 40 . The proposed mechanism for AA improving plant productivity is by improving nutrient use efficiency and increasing photosynthetic rates, improving growth 16,41 .
The results showed moderate and severe water stress conditions increased the EO content, yield, and major constituents, including carvacrol and gamma-terpinene. The production of secondary metabolites such as EOs, alkaloids and flavonoids in medicinal and aromatic plants is considered one of the defense mechanisms to increase plant resistance under stressful conditions 42 . Under drought stress, the massive supply of NADPH + H + in plant cells adversely affects the photosynthesis cycle. Therefore, EOs compounds' productivity using NADPH + H + stored in plant cells balances the NADP+/NADPH+ H+ ratio and improves the photosynthesis cycle 43 . Among different fertilizer treatments, foliar application of AA enormously improved EO quantity and quality by increasing the main EO constituents, including carvacrol, gamma-terpinene, alpha-terpinene and p-cymene. Most EO constituents, such as terpenes composed of an isoprene structure. The isoprene productivity depends on nutrient availability and biomolecules such as ATP, NADPH, and acetyl-CoA 44 . The application of AA enhanced essential oil productivity and major EO constituents by increasing the availability of intermediate compounds, including ATP and NADPH. Similarly, Talaat et al. 45 noted that applying amino acid bioregulators improves the essential oil quantity and quality of Khella (Ammi visnaga L.).
In the present study, seedlings' chlorophyll content (a and b) decreased under moderate and severe water stress conditions. The reduction in the photosynthesis pigments under water deficit stress conditions may be caused by chloroplast breakdown and inhibition of its biosynthesis by enhancing the synthesis of reactive oxygen species (ROS), degradation of chlorophyll precursor, and up-regulation chlorophyllase activity 40 . These conditions increase lipid peroxidation, leading to chlorophyll and photosynthesis pigment degradation 46 . Similar results were found by Rezaei-Chiyaneh et al. 1 study, who noted that the chlorophyll concentration in black cumin (Nigella sativa L.) significantly decreased under drought stress conditions. In contrast, carotenoid content was increased under drought stress conditions due to the higher susceptibility of these compounds to oxidative stress. Therefore, an increase in carotenoid content, as a non-enzymatic antioxidant, under water deficit stress conditions plays a vital role in improving plant tolerance and decreasing the detrimental effects of oxidative stress 47,48 . In addition, chlorophyll (a and b) and carotenoid content increased because of AA foliar application. AA, as an antistress compound, enhances the activity of antioxidant enzymes and proline concentration, leading to decreased ROS harmful effects in the degradation of chlorophylls and carotenoid membranes. Noroozlo et al. 49 stated that the higher chlorophyll and carotenoid content in the lettuce plants could be due to the reduction in chlorophyll degradation and the stimulating impacts of AAs on chlorophyll and carotenoid biosynthesis. Table 6. Correlation coefficient of morpho-physiological characteristics in summer savory (Satureja hortensis L.) PH plant height, LB lateral branch, DY dry matter yield, CD Canopy diameter, EOC essential oil content, EOY essential oil yield, Chla chlorophyll a, Chlb chlorophyll b, Carotenoid carotenoids content, RWC relative water content, Proline proline content, SSC soluble sugars content, CAT catalase, SOD superoxide dismutase, POX peroxidase. ns, * and **non-significant, significant at 5% and 1% probability level, respectively. The results showed that the maximum proline content was achieved from severe water stress (FC50) treatment with AA foliar application. Under drought-stress conditions, plants can alter water relations to maintain cellular functions. For example, plants exhibit osmotic adjustment by synthesizing and accumulating compatible solutes such as free amino acids, sugars, and proline. Also, Hanif et al. 50 noted that proline-based amino acid foliar application and increased proline content would increase osmoprotectants' productivity. Therefore, enhancing proline content through foliar application decreased their negative impacts by stabilizing the membranes, maintaining the osmotic balance and cell turgor, preventing electrolyte leakage, and maintaining the ROS levels in normal ranges (detoxification of ROS) 51 .
RWC is commonly used to quantify plant water status in terms of biochemical and physiological consequences of water stress in plant cells 52 . The results revealed that RWC was decreased under moderate and severe water stress conditions due to increased water loss from savory leaves through transpiration and decreasing water uptake from roots. In this situation, RWC decreased due to reduced leaf water potential 47 . In addition, the AA application strongly increased RWC content in the seedlings. Under drought stress conditions, reducing osmotic pressure by increasing osmotic adjustment such as SSC and proline in the cells helps maintain leaves' turgor pressure. The application of AA enhanced RWC content under stressful conditions through increasing osmotic adjustment and membrane integrity 53 .
CAT, SOD and POX activity were enhanced under severe water stress treated with AA. Under water stress conditions, the antioxidant enzyme activity plays a vital role in eliminating ROS compounds such as superoxide and hydrogen peroxide (H 2 O 2 ). The higher antioxidant enzyme activity reduces ROS compounds in the cells, reducing membrane damage and balancing the performance of cellular activities such as photosynthesis 54 . Also, increased antioxidant activity due to AA foliar application could be attributed to higher nitrogen metabolizing Figure 5. Heatmap clustering of irrigation regime (FC100, 100% field capacity; FC75, 75% field capacity; FC50, 50% field capacity) and fertilizer application (C control, HA humic acid, B bacterial, AA amino acid) sources based on morpho-physiological characteristics in summer savory (Satureja hortensis L.). The key color bar indicates standardized mean values (dark red indicates relatively low mean values; dark blue indicates relatively high mean values).

Scientific Reports
| (2023) 13:720 | https://doi.org/10.1038/s41598-022-27338-w www.nature.com/scientificreports/ enzyme activity because of amino acid availability 16 . In a study, Shafiq et al. 17 reported that a higher proline concentration under drought stress conditions protects macromolecules and enzymes from oxidative damage and increases the activities of the enzymes. Therefore, it can be concluded that AA foliar application can enhance plant protection against drought stress by enhancing organic osmolytes such as proline, SSC, and antioxidant enzyme activity.

Conclusion
Our study revealed that water stress negatively affects savory seedlings, reducing dry matter production by 11% and 33% under moderate and severe water deficit stress. However, foliar application of amino acid enhanced dry matter yield, essential oil content and savory yield by 22%, 31% and 57%, compared with the treatment with no fertilization. Foliar application of amino acid improved savory essential oil quality by increasing essential oil components, including carvacrol, gamma-terpinene, alpha-terpinene and p-cymene. We conclude that the foliar application of amino acid could effectively increase plant tolerance against water deficit stress while improving the essential oil quantity and quality of medicinal and aromatic plants.

Data availability
The authors confirm that the data supporting the findings of this study are available within the article.